Monthly Archives: July 2013

A ‘tail’ to tell – Lakey lab discovery could lead to a new class of antibiotics

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Dr Chris Johnson

“Most of us know that we should wash our hands after being around animals but do most of us know the reasons why? As a researcher who spends most of his time in the lab killing E. coli, using E. coli specific antibiotics I should be well aware of the dangers of this often underestimated Gram-negative bacteria. However when my youngest daughter contracted E. coli O157 after visiting an agricultural show in Scotland in 2011 (even though we had followed the hand washing procedure), I realised that I did not appreciate how nasty this bacteria can be. Thankfully after 6 weeks in hospital including a lengthy stretch on dialysis my daughter made a full recovery but not everyone is so lucky.”

Professor Jeremy Lakey

The urgent need to develop new drugs to target pathogenic bacteria has been a theme of the ICaMB blog since its inception in early 2013.  However, these words from Dr Chris Johnson, a postdoc in Jeremy Lakey’s laboratory, bring home the seriousness of the problem.  Fortunately, Chris is in a position to do something about this.

Many of you will have realised that last week saw another potential breakthrough in ICaMB’s ongoing ‘War on bacteria’ (and here, here and here).   This time the PI making the news, with data demonstrating the possibility of a whole new class of antibiotics, was Professor Jeremy Lakey.  And when we say making the news, we mean that literally. The ITV interview of Jeremy and his team can be viewed here

This story has made news around the world and has been featured in newspapers in Australia and India (and here), as well as closer to home.  Here is the official university press release.

So what’s going on?  If you want to read the primary paper it is here

The unstructured domain of colicin N kills Escherichia coli. Mol Micro 89:84-95

Chris Johnson, who was the lead author on this manuscript explains:

“E. coli produces protein antibiotics called colicins which are used to kill E. coli and closely  related bacteria in the eternal bacterial arms race. In order to further understand how one of these, colicin N (ColN), works, we dissected the protein into its individual domains to see how each part behaved in isolation. Quite by chance we found that part of the protein, THE TAIL, was actually toxic to them. Although far less efficient than the entire ColN molecule, it remains specific for E. coli and furthermore, the specificity is housed within an intrinsically unfolded domain (a domain which has no defined 3D structure). Although this is a very basic discovery in its early stages, it allows us to appreciate novel mechanisms to kill E. coli.” (see the full version of this at the bottom of the page)

Some more about Jeremy

Any of you who were bioscience undergraduates in Newcastle will know Jeremy Lakey from his famous recreations of protein structure using party balloons.  Others may know him for co-founding Orla Protein technologies.  Some of us know him as a man who will always buy his round in the pub.  All of us know him as a great scientist and colleague.  Furthermore, Jeremy is a leading supporter of Leading Edge and recently a group of 6 Year 9 school pupils from St Cuthberts RC School looked at how ColN acts against E.coli when you start changing the amount of salt they are grown in. As many ICaMB scientists may know Jeremy also runs a workshop with Ponteland Community High School to explore bacterial shape and their surfaces using LEGO.

But did any of us think that one day Jeremy Lakey may SAVE THE WORLD from antibiotic resistant bacteria? Possibly.

The detailed science

Most Gram- negative bacteria produce protein antibiotics which are used as weapons in the battle between competing populations of bacteria.  E. coli produces protein antibiotics called colicins which are use to kill E. coli and closely related bacteria.  Once colicins are released into the extracellular milleu they dock onto their targets via specific outer-membrane receptors and then seek out an internal, periplasmic, binding partner (the Tol or Ton proteins) which helps them translocate into the cell.  We study colicin N (ColN) which comprises of an intrinsically unfolded N-terminal translocation (T) domain, involved in TolA and OmpF binding.  Its central receptor binding (R) domain binds lipopolysaccharide whilst its C-terminal 200 amino acids define the cytotoxic pore-forming (P) domain. This latter feature is common to all pore-forming colicins and forms a channel in the inner-membrane causing K+ release and cell death.  Other colicins have C-terminal domains which display cytotoxic activities that include DNAase or RNase activity.  Irrespective of the particular cytotoxic activity, all colicins are comprised of three domains (T-R-P) and it was assumed that the sole role of the T and R domains was to deliver the cytotoxic C-terminal domain across the outer-membrane.

In order to investigate the mechanism of ColN activity we dissected the protein into its individual domains.  We were attempting to block the toxic activity of full length ColN by pre- incubating E. coli cells with the intrinsically unfolded T-domain.  The rationale behind the experiment was that we could block all the available receptor sites on the E. coli target cells by saturating with T-domain, such that when the full length ColN was added to cells it would be non-toxic, as all the essential receptor binding sites would be already sequestered.  However rather than protecting the cells, T-domain was found to be toxic and like full length ColN provoked K+ efflux.   Although less efficient than full length ColN, T-domain is strictly dependent upon the same receptor proteins, OmpF and TolA for killing.  Since these receptors are only found in E. coli-like bacteria, T-domain displays the unusual combination of a generic killing mechanism coupled with extreme specificity housed within an intrinsically unfolded domain.

Links

Jeremy Lakey’s University home page: http://www.ncl.ac.uk/camb/staff/profile/jeremy.lakey

Follow Jeremy Lakey on Twitter: https://twitter.com/JeremyLakey

ICaMB: http://www.ncl.ac.uk/camb/

The Centre for Bacterial cell Biology: http://www.ncl.ac.uk/cbcb/

The official Newcastle University press release: http://www.ncl.ac.uk/press.office/press.release/item/chance-finding-could-lead-to-new-antibiotics

Link to ITV story:

http://www.itv.com/news/tyne-tees/story/2013-07-05/breakthrough-in-combatting-bacterial-infection/

The ‘Australian’ story: http://www.theaustralian.com.au/news/breaking-news/tail-could-be-used-for-new-drugs/story-fn3dxix6-1226674131684

The ‘Times of India’ Story: http://articles.timesofindia.indiatimes.com/2013-07-06/science/40406695_1_escherichia-coli-e-coli-protein

The Northern Echo story: http://www.thenorthernecho.co.uk/news/10524003.Scientists__chance_find_may_develop_new_generation_of_antibiotics/

Jeremy’s company, Orla Proteins: http://www.orlaproteins.com/about-orla/the-board.aspx

 

Leading the Way… in Protein Structure

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By Kevin Waldron

This week, ICaMB welcomed the Leading the Way winners into our labs for an exciting day of science. As you may remember from our previous post a couple of weeks ago, Leading the Way was ICaMB and Leading Edge’s collaborative pilot scheme to take some of ICaMB’s great science (and early career scientists) into a local school, George Stephenson High School in Killingworth. That week was a great success, inspiring all of its participants: students, teachers and ICaMB members alike.

The overall winners during the week in GSHS were the AU team, made up of Lucy Hainsworth, Libby Macpherson, Rebecca Brown, Lauren Rhodes, Abbey Wrightson, Kimberley Stoker, Sophie Anson, Connor Little, Nathan Clapperton. AU designed an outstanding poster to illustrate how the prion protein represents a biomarker of mad cow disease (or, more scientifically, variant Creutzfeld-Jakob disease, vCJD), how the structure of this protein changes from the ‘normal’ form to the ‘abnormal’, disease-causing form, and how knowing the structure of the prion protein can enable us to design a diagnostic test.

AU’s prize was to spend a day in an ICaMB laboratory, learning about how we determine the structure of a protein, with the members of the judging panel, Dave Bolam, Paula Salgado and myself.

After a brief welcome and introduction, we kitted our guests out in fetching lab coats, supplied them with ‘Leading the Way’ lab books and got started.

Showing the kids how to plate cells on a petri dish

 

First, the kids tried their hands at microbiology with the Waldron lab, streaking E. coli cells onto agar plates and then picking colonies to inoculate cultures for recombinant protein production.

 

 

Practising to become a PhD student, staring at a pouring column…

Next, Dave Bolam and his team demonstrated how a His-tagged protein can be purified using affinity chromatography, and then the kids loaded each of their protein samples on SDS-PAGE gels. Remarkably, all of the students successfully purified their target protein, though it’s worth noting that this was not actually the prion protein, PrPC (Imagine the risk assessment!).

 

“Here, let me help you” says Dave.

 

“It was great fun spending time with enthusiastic kids and giving them a flavour of what we do. It reminds you why you do science in the first place“, says Dave after taking part in this type of activity for the first time.

“We’re doing science now, Miss!” – shouted one of the students

 

 

The day finished with a demonstration of protein crystallisation with Paula Salgado and Will Stanley of the Structural Biology Laboratory. The students attempted to crystallise lysozyme (with mixed success), and then observed protein crystals under the microscope.

“It’s always great to share our love for science with young minds and see them get really excited about carrying out the experiments we do routinely. It’s a breath of fresh air in the lab. Hopefully we’ve given them an experience to remember, as well as a better understanding of research in a biomedical institute.” commented Paula at the end of the day.

 

Although this was a high-paced tutorial in protein production and structure determination – a process that usually takes at least several weeks, and in some extreme cases an entire career – the students received a hands-on demonstration of some real-life research techniques. We all hope that this experience, even at such an early age, might just implant the idea of a future in science for some of these young people.

But from my own perspective, I can say that their enthusiasm has been infectious (no pun intended), and is a timely reminder of why I got into science in the first place – because at school I always found science classes more interesting than any others. I would have loved such an opportunity when I was that age.

Thanks go to Phil Aldridge, ICaMB’s Leading the Way coordinator, all of the members of the Waldron, Salgado and Bolam labs who helped out during the visit, the staff of George Stephenson High, and most of all to the members of the AU team, for making the day a success.

 

George Stephenson High School http://www.gshs.org.uk/home